This page describes how to analyze fluorescence intensity (ΔF/F₀) from a series of images using ImageJ and Excel. While semi-automated custom tools are often used to facilitate rapid analysis of fluorescence data, there is great value in understanding how to realize these analyses using standard and commonly-available software.

Measure ROI Fluorescence

• Install ImageJ (Fiji)
• Drag/drop a folder of images onto ImageJ to open all images as a stack
• Create a maximum projection (of either channel) to make drawing of ROIs easy. Image > Stack > Z project and select Average (not maximum projection!)
  • do all ROI creation on the projection. You can later use these same ROIs on the original data.
• Click analyze > tools > ROI manager (ROI means “region of interest”)
• Use the “freehand sections” tool (as opposed to the square or circle) to outline some cells.
  • Every time you outline a cell, click the add button on the ROI manager.
  • I recommend naming cells as you add them (click the rename button).
  • Be sure to indicate whether the thing is a neuron or astrocyte.
• When all cells have been outlined, highlight them all (with shift-click or control-click) and save the ROIs by clicking more > save.
  • once ROIs have been saved, you can load the same ROIs on the red stack, green stack, or a projection image
• Deterime what data to analze by clicking analyze > set measurements
  • I recommend selecting ONLY “Mean gray value”
• Analyze the data in the ROI manager by clicking more > multi measure
• In the results window, click file > save as and call it something
  • Excel 2016 may not be able to open the .xls file. You may have to rename it to .tsv (short for tab-separated values) and drag/drop it into a blank excel worksheet.
• Once the data is in excel, add a “time” column and populate its values appropriately

Calculate Single-Channel ΔF/F₀

Single-channel fluorescence experiments report the change in fluorescence (ΔF) normalized to its baseline level (F₀). In this example F is measured for each ROI in every frame. The following steps are performed for each ROI.

• Calculate F₀ as the mean fluorescence intensity during the baseline region

• Calculate ΔF by subtracting F₀ from every F value

• Calculate ΔF/F₀ by dividing ΔF values by F₀

Calculate Ratiometric ΔF/F

The following describes how to measure ratiometric fluorescence from a series of 2D multi-channel images. See Ratiometric Linescan Analysis with ImageJ and Excel for information specific to analyzing ratiometric linescan images.

Two-channel fluorescence experiments report ΔF/F as the change in one fluorophore relative to another. In this example we will use G to represent a calcium-sensitive fluorophore and report the change in its fluorescence relative to a calcium-insensitive fluorophore R. In this example G/R is measured for each ROI in every time point. The following steps are performed for each ROI.

In this case F represents G/R so ΔF/F is: [Δ(G/R)]/(G/R)₀

• Calculate (G/R)₀ as the mean ratio of fluorescence intensity during the baseline region

• Calculate Δ(G/R) by subtracting (G/R)₀ from every (G/R) value

• Calculate ΔF/F by dividing Δ(G/R) values by (G/R)₀